Method for analysis of haemostatic activity

ABSTRACT

This invention relates to a method of analyzing coagulative, fibrinolytic or haemostatic activity in, especially, blood or plasma from mammals, particularly humans. The method comprises bringing a sample, in vitro, into contact with fixed and preferably from their growth support detached endothelial cells or outer membranes of such cells, and detecting the resulting coagulated material and, in some cases, lyzed coagulated material. Reagents are also described that are based on the endothelial cells and their outer membranes. A kit for performing such a method including the claimed reagent is also described.

FIELD OF INVENTION

The present invention pertains, in general, to the field of haemostasisor to the study of the haemostatic system of mammals, in particularhumans. More specifically, it pertains to a new, simple and therebyclinically useful analytical method which allows evaluation of eithercoagulative or fibrinolytic activity or even total haemostatic activityin a biological fluid. Primarily, this means analysis, in a simple yetreproducible and reliable manner, of blood or blood plasma in order todetect haemophilia and/or thrombophilia. The technique according to theinvention is based on contact between the biological fluid and a reagentwhich effects the coagulative process, after which coagulated materialis detected according to prior art principles. The novelty in relationto the invention is the utilization of a reagent that highly resemblesthe factors that in vivo effect coagulation in a mammalian individual,which gives better possibilities than the prior art to obtain a totalpicture of the haemostatic activity.

The invention also pertains to a kit for performing this method.

BACKGROUND OF THE INVENTION

Circulatory disturbances can lead to illness with bleedings or thrombi,clinical conditions which are denoted haemophilia or thrombophilia,respectively. Such disturbances may result from perturbations in theability of the blood to coagulate or to dissolve coagulated material orfrom perturbations of the balance between these. Coagulum formation andcoagulum dissolution are both precisely regulated processes, both ofwhich can be regarded as composed of a driving and a restraining part.The coagulum-forming process is called coagulation and is hence abalance between pro-coagulative and anti-coagulative activities.Analogously, the coagulum dissolving process is called fibrinolysis anddisplays a pro-fibrinolytic and an anti-fibrinolytic part. The balancebetween coagulation and fibrinolysis is denoted haemostasis, which thuscomprises all processes that prevent leakage of blood from thecirculatory tract and which keep these open. The two conceptscirculatory disturbances and haemostatic disturbances are in thiscontext practically synonymous.

The understanding of haemophilia and the development of effectivetreatments for this condition has been strongly dependent on simpleglobal functional laboratory methods, which have revealed alteredcoagulative properties of blood and blood plasma from the individuals inquestion. The principle of these methods is to add, to blood or bloodplasma, reagents that trigger coagulation, and to register the reactiontime necessary for a certain coagulum formation. Abnormally slow or weakcoagulum formation has been characteristic of haemophilia, and medicaltreatments which have normalized the analytically determined values haveproved to be effective in the alleviation of the clinical symptoms ofthe disease.

According to the above described view on hemostasis, the outlined priorart global functional laboratory methods for the characterizaton ofcoagulative activity are primarily sensitive to pro-coagulativedisturbances. The methods have, however, appeared to be fairlysatisfactory for the diagnosis of haemophilia because the haemostaticdisturbances involved are often localized precisely to thepro-coagulative part of haemostasis. However, the prior art globalmethods cannot reveal disturbances in all haemophiliacs, and they arepractically of no use for thrombophilia. This may be due to the factthat the methods do not give any indication, or only slight indication,of disturbances in the anti-coagulative part of the coagulation process,and that they lack sensitivity to fibrinolytic disturbances. The lack ofor the need of a more comprehensive method, useful in practice, foranalyzing or detecting coagulative, fibrinolytic and haemostaticproperties of blood and blood plasma, is therefore obvious. A simple andespecially a more reliable method relating to global haemostasis woulddoubtless result in improved diagnostics, improved treatment and moreefficient development of methods of treatment, especially ofthrombophilia. The present invention provides an improved globallaboratory method of diagnostic analysis of coagulative, fibrinolytic orhaemostatic properties of blood and blood plasma. Moreover, the methodis of such nature that it is directly applicable to correspondinganalysis of other biological fluids wherein such activities are present,for example synovial fluid and brain liquor.

DESCRIPTION OF THE INVENTION

The present invention thus provides a method of analysis of biologicalfluid from a mammalian individual, in particular human, for the purposeof determining or analyzing coagulative activity or fibrinolyticactivity in a mammalian individual or even the total or globalhaemostatic activity in said fluid. In other words, it is possible,according to the invention, to identify individuals having dispositionfor haemophilia as well as individuals having disposition forthrombophilia by using one and the same method of analysis. Moreover,this can be achieved according to the invention in a most simple mannerby performing the analysis in in vitro on very small amouts of sample ofthe biological fluid in question. This makes the method according to theinvention particularly well suited for laboratory diagnostic use.

The method according to the invention is based on the surprisingdiscovery that devitalized endothelial cells or devitalized and fromtheir support liberated endothelial cells trigger coagulation in abiological fluid, especially in blood and blood plasma, in such a mannerthat haemostatic disturbances typical for both haemophilia andthrombophilia can be detected. Moreover, it has been discovered that thecoagulated material, which is formed when the biological fluid iscontacted with devitalized endothelial cells, can serve as a substratefor the fibrinolytic activity of the biological fluid which thereforecan be analyzed and characterized according to the invention. This inturn makes it possible, as will be detailed below, to analyze andcharacterize a quantity that can be identified with haemostaticactivity. Thus, it has been discovered that devitalized endothelialcells, and devitalized and from there their support liberatedendothelial cells, posses considerable diagnostic potential with regardto haemostatic disturbances.

Nothing has hitherto been revealed about the properties of endothelialcells and their interactions with biological fluids, particularly bloodand blood plasma, (see Stern et al. 1985, Proc. Natl. Acad. Sci. 82,2523-2527 and Kirchhofer et al. J. Clin. Invest. 93, 2073-2082) thatwould initiate the use of these cells in in vitro laboratory diagnosticcontext. The generally accepted view that endothelial cells are stronglyanti-coagulative should refrain a man skilled in the art from attemptingto use endothelial cells in such diagnostic contexts where coagulativeor fibrinolytic or haemostatic properties of a sample are to beanalyzed. At least it can be established that the diagnostic potentialof endothelial cells in this context has neither been concieved norobserved. As example of prior art, EP-A-538 951 can be mentioned.Therein, the difference between tissue factor and thromboplastinactivity between cells from extra-cellular tissue and endothelial cellsis mentioned, i.e. endothelial cells have been considered useless in thecontext contemplated by the present invention.

Nevertheless, according to the present invention it is shown thatendothelial cells can be fixed and detached from their support, or viceversa, and that these fixed endothelial cells still posses activities ofsuch nature that they are useful as a main component in reagents foranalysis or characterization of coagulation, fibrinolysis andhaemostasis in a mammalian individual. The nature of the reagent used inthe method of the invention will thereby be very close to the reality inthat coagulation under the influence of a system of co-operating andopposing factors are studied according to the invention in stead of somefactor or a few factors disconnected from their context. The fact thatdetachment of the endothelial cells from the support on which they havebeen cultured or allowed to grow apparently does not harm the, in thiscontext, active membrane structure, allowes the present invention toutilize the effects mentioned, in a particularly advantageous way, inalready existing equipment. For example, the new reagent according tothe invention can be used in regular coagulation instruments as aparticulate suspension or can be used to coat a solid surface toimmobilize the particulate preparation. Still, the method according tothe invention comprises also the case when the endothelial cells usedare not detached from their original place of growth, even though this,in many cases, means that full advantage of the surprising discovery ofthe activity in question of the fixed and detached cells, is notobtained.

The method according to the invention is thus a method of analysis,particularly quantitative analysis, of coagulative, fibrolytic orhaemostatic activity in a biological fluid, especially blood or bloodplasma, from a mammalian individual, in particular human, wherein thecharacteristic feature of the method is contacting a quantity of sampleof said fluid, in vitro, with fixed and preferably from their place ofgrowth detached endothelial cells, or outer membranes of such cells, anddetecting the resulting coagulated material, preferably by measuring thetime required for a certain amount of coagulum to form or by measuringthe amount of coagulum formed during a certain period of time. Bothbefore and after the coagulum-lytic activities of the sample have hadthe opportunity to be expressed, detection of the resulting coagulatedmaterial can be done and, this proves to be more advantageous, theresult of the lytic processes is detected.

More specifically an aspect of the invention is directed to a method ofanalysis, particularly quantitative analysis, of coagulative,fibrinolytic or haemostatic activity in to a biological fluid,especially blood and blood plasma, from a mammalian individual,especially human, wherin a quantity of a sample of biological fluid,especially blood or blood plasma, is brought into contact in vitro withfixed and preferably from their place of growth detached endothelialcells, or outer membranes of such cells, and either

a) the rate of coagulum formation or the time required for a certainamount of coagulum to form is determined, this rate or coagulation timebeing compared with reference values of rates and coagulation times ofnormal individuals, and if this rate or coagulation time is shorter thanthe reference values the sample of biological fluid is likely to comefrom an individual having disposition for thrombosis, whereas if therate or coagulation time is longer than the reference values then thesample of biological fluid is likely to come from an individual havingdisposition for bleeding, or

b) the coagulative processes are disrupted and the amount of coagulumformed during a certain contact time is determined, the amount ofcoagulum being compared with reference values of amounts of coagulum ofnormal individuals, and if the amount of coagulum is smaller than thereference values then the sample of biological fluid is likely to comefrom an individual having disposition for bleeding, whereas if theamount of coagulum is greater than the reference values then the sampleis likely to come from an individual having disposition for thrombosis,or

c) the coagulative processes are disrupted after a certain time and anadditional certain reaction time is allowed, followed by determinationof the amount of dissolved coagulated material, this amount of dissolvedcoagulated material being compared with reference values of dissolvedcoagulated material of normal individuals, and if the dissolved amountof coagulated material is greater than the reference values then thesample of biological fluid is likely to come from an individual havingdisposition for bleeding, whereas if the dissolved amount of coagulatedmaterial is smaller than the reference values then the biological fluidis likely to come from an individual having disposition for thrombosis,or

d) the coagulative processes are disrupted after a certain time and anadditional certain reaction time is allowed, followed by determinationof the residual amount of coagulated material, this amount of residualcoagulum is compared with reference values of amounts of residualcoagulum of normal individuals, and if the amount of residual coagulumis smaller than the reference values then the sample of biological fluidis likely to come from an individual having disposition for bleeding,whereas if the amount of residual coagulum is greater than the referencevalues then the biological fluid is likely to come from an individualhaving disposition for thrombosis.

When the procedural step a) or b) is performed, results pertaining tocoagulative activity are obtained. When the procedural step c) isperformed, results pertaining to fibrinolytic activity are obtained, andwhen the procedural step d) is performed results pertaining tohaemostatic activity are obtained. Alternatively, the fibrinolytic, orcoagulum-lytic, activity can be identified as the difference betweenassay results obtained in b) and d).

In one embodiment of the invention, the amount of sample of thebiological fluid is previously reversibly anti-coagulated blood or bloodplasma, which in connection with the mentioned in in vitro contact isreturned to its coagulation-active state. Thus, for exemple, the fluid,which can be blood or blood plasma, is anti-coagulated with a substancethat binds Ca²⁺ ions, preferably citrate or EDTA, and the return to itscoagulation-active state is accomplished by addition of excess amountsof Ca²⁺ ions. Alternatively, the fluid may be anti-coagulated with acoagulation-inhibiting substance, e.g. hirudin or heparin, and thereturn to the coagulation-active state is accomplished by addition of asubstance which inhibits the effect of said coagulation-inhibitingsubstance, for exemple antibodies that inhibit the activity of hirudinor heparinase, respectively.

Conveniently, the analysis is performed on an amount of sample ofbiological fluid of maximally 1.0 mL, preferably 0.01-1.0 mL. Mostpreferably the amount of sample is maximally 0.2 mL, and the coagulationtime is maximally 15 minutes.

In performing the procedural step b), c) or d), the coagulativeprocesses may be disrupted by the addition of a coagulation-inhibitingsubstance, which preferably is chosen from the group consisting ofhirudin and heparin but also Ca²⁺ ion complexing agents such as citrate,oxalate or EDTA are possible.

In performing the procedural step a) or b), the analysis may beperformed in the presence of a fibrinolysis-inhibiting substance, whichpreferably is chosen from the group of 6amino-hexanoic acid andantibodies against tPA and uPA.

The result of performing the procedural step a), can be obtained bydetecting coagulated material by means of measuring changes in theoptical or rheological properties of the reaction mixture, preferablychanges in light transmission or viscosity.

The results of performing the procedural step b), c) or d) can beobtained by detecting coagulated material by measuring some component ordegradation product of the coagulated material preferably fibrin orthrombocytes. Conveniently, results in this regard are obtained bymeasuring the amount of fibrin via its enzymatic degradation, preferablyby plasmin, to soluble degradation products, which are measured withimmunological technique. So, for exemple, said degradation products maybe measured with an immunological technique which is specific forD-dimer fragments, or the amount of thrombocytes associated with fibrinmay be measure by flow cytometry after enzymatic degradation of thefibrin.

Another aspect of the invention is directed to a reagent for theanalysis of coagulative, fibrinolytic or haemostatic activity of abiological fluid, especially blood or blood plasma, from a mammalianindividual, especially human, which reagent is composed of suspended orsupport-attached endothelial cells or outer membranes from such cells.

In an embodiment of of this aspect of the invention, the endothelialcells or their outer membranes are from an individual of the samespecies as the individual whose biological fluid is to be analyzed,preferably from the same individual and most preferably from the part ofthe body which is subjected to said analysis.

In another embodiment, the endothelial cells or their outer membranesare immobilized on a solid phase. In order to stabilize the activesurface structures, polyethylene ethers or polysaccharides can becovalently bound to the outer membranes of the endothelial cells.

Yet another aspect of the invention is directed to a kit for performinga method of analysis of coagulative, fibrinolytic or hemostatic activityin a biological fluid, especially blood or blood plasma, from amammalian individual, especially human, which kit comprises, as acoagulation reagent, a reagent according to the invention andinstructions for performing the method.

According to the invention, it has been shown that devitalized (othercommonly used denotation; fixed) or devitalized and detached from theirsupport (other commonly used denotation; suspended) endothelial cellscan be used to characterize coagulative, fibrinolyfic and hemostaticactivity of biological fluids, especially blood and blood plasma, andthat this can be accomplished in a diagnostically valuable manner, i.e.so that individuals with circulatory diseases can be differentiated fromnormal individuals. Functions typical for the living endothelial cell,such as excretion, endocytosis and modifications of outer membranestructures, re obviously not necessary in order for endothelial cells totrigger activities in biological fluids that make meaningful diagnosispossible. From this it is obvious th also isolated outer membranes fromendothelial cells can be used in practising the invention. Thus, the useof such outer membranes does not represent any deviation from the spiritof the invention, and in this context even synthetically manufacturedstructures characteristic for the surface of endothelial cells may becontemplated. In other words, the expression "outer membranes of suchcells" should interpreted broadly and it encompasses all attempts to(also synthetically) arrive at membrane-like structure that triggers thesame or similar activities in biological fluids, especially blood andblood plasma, as fixed or fixed and suspended endothelial cells.

The fixed endothelial cells used in the method according to theinvention, can be obtained from different sources and can be prepared indifferent ways without deviating from the original idea of theinvention. Endothelial cells can, for example, be obtained from culturedcells which in accordance with known methodology have been detached fromthe interior surface of umbilical cord veins. Placenta is anotherpossible source of endothelial cells as are established cell lines oftransformed endothelial cells. It should be understood that an effectivepreparation according to the invention, of fixed or fixed and from theirsupport detached endothelial cells, does not necessarily need to betotally homogeneous. It is sufficient that a main constituent of a cellpreparation according to the invention is endothelial cells.

Preferably such endothelial cells are chosen that are from an individualof the same species as the individual from whom the biological fluid isobtained. More preferably the endothelial cells are from the sameindividual and most preferably from the part of the body which issubject to analysis according to the invention. Choosing endothelialcells from the same individual as from whom the sample of biologicalfluid is obtained would allow detection also of circulatory disturbancesthat stem from abnormalities of this individual's endothelial cellstructures that together with this individual's biological fluid expressdisturbances in coagulative, fibrinolytic and/or hemostatic activity.

In the preparation of endothelial cells from cultures of such cells, itis possible to detach these cells from the support on which they grow,so that a mainly monodisperse suspension is obtained. The cells can, forinstance, be detached by citrate or an other substance that binds Ca²⁺ions and can thereafter be fixed. Other methods for detaching theendothelial cells, e.g. by use of EDTA, are also within the scope of theinvention. If the cells are detached from their support with the aid ofenzymes, it may be necessary to culture the cells for some time beforethey are fixed, in order to allow necessary surface structures toreform. Different methods for fixation of cells are certainly previouslyknown in the prior art.

These methods are applicable also in the present case, but preferably inthe method according to the invention the endothelial cells aredevitalised or fixed with substances chosen from the group of formalinand STF™ fixation solution.

The invention is nether restricted to the case where the cells are atfirst fixed and thereafter detached from their support. The reversecourse of action is fully possible within the scope of the invention.

Specific analysis of coagulative, fibrinolyfic or haemostatic activityis possible according to the invention but may require additions ofcoagulation-inhibiting or fibrinolysis-inhibiting substance, which willbe described in detail below.

As preferred examples of coagulation-inhibiting substances hirudin andheparin can be mentioned, and as preferred examples offibrinolysis-inhibiting substances 6-amino-hexanoic acid and antibodiesagainst tPA and uPA can be mentioned.

A considerable advantage with regard to the use of fixed endothelialcells according to the invention, is that these can be utilized in theform of a suspension, preferably a water-based suspension, which enablesthe use of equipment for detection of coagulation existing on themarket. As examples of such equipment, coagulation instruments operatingwith liquids or suspensions and wherein detection of coagulum formationmost often is performed photometrically, may be mentioned.

The in vitro contact between the fixed endothelial cells, or outermembranes, and the biological solution is, however, not restricted tothe use of suspensions but can be accomplished by using other principlesknown per se. For example, the contact can be accomplished withendothelial cells or outer membranes immobilized on a solid phasepreferably of the micro test plate type.

Immobilization on the solid phase can preferably be accomplished viabinding of biotin to the endothelial cells or outer membranes, andthereafter coupling to the surface of the solid phase by means ofbiotin-binding substances, preferably streptavidin.

According to yet another preferred embodiment of the method according tothe invention, preparations of endothelial cells, preferably suspended,or corresponding outer membranes, with increased stability andhomogeneity are obtained in case the endothelial cells are treated withreactive compounds that covalently bind hydrophilic substances to theouter membrane structures of the cells. As preferred examples of suchstability- and homogeneity-improving compounds polyethylene ethers andpolysaccharides may be mentioned. Thus, such improved stability andhomogeneity are extremely valuable in a method of the present kind,wherein the analysis is dependent on reasonably stable reagents.

Coagulated material that results from performing the method according toinvention, is preferably detected by means of measuring changes in theoptical or Theological properties of the reaction mixture, especially incase the use of cells in suspension is involved. Preferably, thisconcerns changes in light transmission and viscosity, respectively.

Coagulated material can according to another embodiment be detected bymeans of measuring changes in the interactions of the reaction mixturewith magnetic, electric or electromagnetic fields. This methodology isparticularly suitable for working with cells immobilised on a solidphase.

Coagulated material is preferably detected via the components itcontains or is made up of, particularly fibrin and thrombocytes.

The amount of fibrin can preferably be measured by enzymatic degradationof the same, preferably by plasmin, to soluble degradation productswhich are measured with immunological technique. A preferred suchtechnique is a technique specific for D-dimer fragments.

Another preferred embodiment of the invention measures the amount ofthrombocytes associated to the thrombocytes, for example by flowcytometry. This can be accomplished after the coagulum has beenenzymatically degraded, i.e. has been lyzed.

As indicated above, the method according to the invention is primarilyintended for analysis of blood and blood plasma but is in no wayrestricted to this and can be applied in any instance where activitiesof the corresponding type exist.

For the analysis to be useful in practice in clinical contexts, thisimplies that, in the method, the use is made of previously reversiblyanti-coagulated blood or blood plasma which is returned to thecoagulation-active state in dose connection with the contacting in vitrowhich, according to the invention, is performed with fixed endothelialcells or corresponding active components of these.

One preferred embodiment involves blood or blood plasma that has beenanti-coagulated with substances that bind Ca²⁺ ions, preferably citrateor EDTA, and that the return to the coagulation-active state isaccomplished by addition of Ca²⁺ ions in excess.

Another interesting embodiment requires that the blood or blood plasmais anti-coagulated with a coagulation-inhibiting substances of hirudinor heparin type and that the return to the coagulation-active state isaccomplished by addition of agents that inhibit the effects of theanti-coagulative substances in question, preferably antibodies, thatneutralize the activity of hirudin, and heparinase, respectively, thatdegrades heparin into inactive saccharides.

For a method of clinical laboratory utility it is moreover required thatit must be operable on small quantities of sample. This requirement isfulfilled in the present case, since the method according to theinvention can be performed on such small amounts of sample. According toone preferred embodiment of the invention this means that the analyticalmethod is performed on an amount of sample that is smaller than 1.0 mL,preferably 0.01-1.0 mL.

For laboratory diagnostic utility, especially on larger scale, it isfurthermore required that the analytical method should not beexcessively extended in time. Coagulative activity may according to theinvention be determined in less than 15 minutes and fibrinolytic andhaemostatic activity within 90 minutes.

The invention also pertains to a kit for performing an analytical methodof the type defined above, the characterizing feature of said kit beingthat it contains a coagulation reagent comprising the above mentionedendothelial cells or outer membranes of these, and instructionsdisclosing how the method is to be performed.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be further illustrated below by means of a number ofconcrete practical examples, which however do not in any way restrictthe scope of the invention. Before these are presented in detail, ashort background of the methods and materials used will be given asfollows.

Methods which today are commonly used for laboratory detection ofcoagulative properties of blood and blood plasma are denoted activatedprothrombin time, APTT, prothrombin time, PT, and prothrombin complextime, PTK. These methods are based on the use of one or two reagents,which are mixed with the sample of blood or blood plasma, which isthereby coagulated. The speed with which the reaction mixture coagulatesconstitutes the assay response. Often time in question is measured fromthe moment of reagent addition up to a certain coagulum formation. Thisis detected by measuring a certain change in the optical properties ofthe reaction mixture or a certain change in its rheological properties.

The analyses are often performed on samples that are anti-coagulated byaddition of citrate or other substances that bind Ca²⁺ ions, which arenecessary for the coagulation processes. The reagents contain Ca²⁺ ionswhich restore the coagulative ability of the samples. Ifnon-anti-coagulated samples are to be analyzed, the reagents need notcontain Ca²⁺ ions as sufficient amounts of these are present in theblood sample.

The APTT reagent contains silica particles, and the PT- and PTK-reagentscontain tissue factor preparations. Both of these reagents containphospholipids.

With the above described generally used methods, blood and blood plasmafrom individuals without circulatory disturbances display normalcoagulation times while samples from haemophiliacs display prolongedcoagulation times. Samples from thrombophiliacs, almost withoutexception, display normal coagulation times.

According to one aspect of the method according to the invention,Individuals without circulatory disturbances display normal coagulationtimes, while individuals with haemophilia often display abnormally longcoagulation times and individuals with thrombophilia in many casesdisplay abnormally short coagulation times.

In one embodiment of the procedure according to the invention, a reagentcomposed of fixed endothelial cells suspended in aqueous solutioncontaining Ca²⁺ ions is used. When this reagent is mixed with blood orblood plasma anti-coagulated with citrate, longer than normalcoagulation times are obtained for most haemophiliacs and shorter thannormal for many thrombophiliacs. The coagulation times are a measure ofthe coagulation activity of the blood plasmas. With an identical methodcoagulation activity can in a similarly advantageous manner bedetermined for blood and other biological fluid such as synovial fluidand liquor.

Coagulative activity of anti-coagulated blood, blood plasma or otherbiological fluid can be analyzed and characterized also with otherembodiments of the method according to the invention. According to onesuch embodiment, an anti-coagulated sample of the biological fluid isadded to small containers, e.g. wells of micro test plates, the surfaceof which carry fixed endothelial cells. Once the sample has beenreturned to a coagulative state by the addition of substances thatneutralize the anti-coagulative effect, the coagulative processes areallowed to proceed for a certain time after which they are halted byaddition of coagulation-inhibiting substances such as hirudin orheparin. The amount of coagulated material is thereafter measured.Either the amount of fibrin or the number of thrombocytes in thecoagulated material is measured. The amount of fibrin can be measured byenzymatically degrading this to soluble degradation products, followedby measurement of the amount of these by immunological techniques. Thenumber of thrombocytes in the coagulated material can be determined,e.g. with flow cytometry, after the thrombocytes have been liberatedfrom the coagulated material by enzymatic degradation of the same. Itshould be mentioned that, if the fibrinolytic activity of the sample isconsiderable, disturbances of the analysis of coagulative activity canbe avoided by addition of fibrinolysis-inhibiting substances to thesample prior to analysis. The fixed endothelial cells on the innersurface of the reaction vessel can either be cultured on this site or,which is more practical and better exploits the possibility of theinvention, be immobilized thereon from preparations of fixed and fromtheir support detached endothelial cells.

The invention also allows for analysis of the fibrinolytic activity ofthe sample solution. For this purpose the coagulated material is firstallowed to form under a certain time. Thereafter, the reaction isstopped by addition of coagulation-inhibiting substances, e.g. EDTA,hirudin or heparin, after which sufficient time is allowed for theresults of the fibrinolytic activity of the sample to become measurableas increase of the amount of fibrin degradation products. It is pointedout that the fibrinolytic activity in a sample of biological fluid oftenis unstable. This can be caused by inhibitory activities, particularlyplasminogen activator inhibitor type I (PAI-1), and can require specialsample collection and sample handling techniques. Thus, for exemple, thepH of the sample can be immediately reduced to between 5.8 and 6.0 (seeRanby et al 1989, Thromb Haemostas 62, 917-922) to prevent reactionbetween tPA and PAI-1. For analysis according to the invention of suchacidified samples, sufficient amounts of pH-raising substances may beadded in order to avoid hampering of the coagulative and fibrinolyticprocesses. Correct assessment of the fibrinolytic activity may alsorequire addition of substances that prevent said reaction after the pHhas been neutralized. An example of such a substance is inhibitoryantibodies towards PAI-1.

An analytical method that resembles the latter application of theinvention enables an assay response that can be identified withhaemostatic activity. Hereby the coagulative activity is first allowedto express for a certain time during which an amount of coagulatedmaterial in proportion to the coagulative activity is formed, whereuponthe coagulative processes are stopped by the addition of acoagulation-inhibiting substance. Thereafter, the fibrinolytic activityis allowed to act under a sufficient time for creation of a measurablereduction of the amount of coagulated material. The amount of coagulatedmaterial that then remains constitutes a measure of the balance betweencoagulation and fibrinolysis, i.e. the hemostatic activity. The amountof coagulated material is measured as the amount of fibrin and/or as thenumber of coagulum-associated thrombocytes, as previously described.

The method according to one embodiment of the present invention is basedon the use of reagents that are mixed with samples in principally thesame manner as in the analysis according to the methods of APTT, PT andPTK. The coagulation times are registered in the same manner as in thesemethods.

EXAMPLES

Materials

Human endothelial cells from umbilical cord were prepared from 30 to 40cm long umbilical cords from full term foetus. The umbilical cords wereobtained from the maternity ward, the University Hospital of Linkoping,Sweden. Prior to preparation, the umbilical cords were stored for notmore than 24 hours at 2-4° C. in sterile PBS (phosphate buffered saline)with penicillin and gentamycin. During preparation the umbilical cordvein was, rinsed with about 50 mL of sterile PBS containing penicillinand gentamycin, filled with cell culture medium A (Dulbeccos MinimalEssential Medium, 1% non-essential amino-acids, 2% 200 mM L-glutamine,1.2% 100 mM/20 IU/mL oxalo-acetic acid/insulin, 12% foetal calf serumand 0.1% penicillin-streptomycin) containing 500 mg/L collagenase,closed at both ends with Peans and incubated for 15 minutes at 37° C.,whereupon it was massaged for 2 minutes. The contents of umbilical veinincluding 25 mL of rinse (medium A) were transferred to a 50 mL testtube, which was centrifuged for 5 minutes at about 500 xg forsedimentation of the endothelial cells. The supernatant was discardedand the endothelial cells suspended in 10 mL of medium A. Theendothelial cells were again allowed to sediment by centrifugation for 5minutes at 500 xg, after which the supernatant was discarded and thecells were suspended in 5 mL of medium A and transferred to a 23 cm²cell culture flask with gelatin treated bottom surface. This primaryculture was incubated for 2 to 4 days at 37° C. in an atmospherecontaining 5% CO₂. When the cells had developed into a confluentmono-layer, verified microscopically, they were passaged. The passageindicates that the cell culture medium was discarded and the cellsdetached from their support by treatment with minimal amount of 10 g/Ltrypsin dissolved in 0.15 M NaCl and 5 mM EDTA. The detached endothelialcells were suspended in 10 mL of medium A and seeded into two cellculture flasks each with a gelatin-treated bottom surface of about 23cm². At confluence, after 2 to 4 days of culture, the cells werepassaged a second time and seeded out in four cell culture flasks withgelatin-treated bottom surface of 23 cm² each. For some experiments, theendothelial cells were seeded out in about 50 micro test plate wellswith gelatine treated bottom surfaces. When these cells, in thefollowing called HUVEC (human umbilical vein endothelial cells), within2 to 4 days formed a confluent mono-layer, they were used in theexamples below.

Gelatin treatment of cell culture flasks and micro test plate wells wasperformed in such manner that the bottom surfaces were covered with 0.2%gelatin in water, incubated for 15 minutes, emptied of excess andallowed to air dry.

For Example 1 were used: suspended devitalized (fixed) endothelialcells, that were obtained by washing the HUVEC cells in one cell cultureflask with PBS and detaching them from their supporting place of growthby 15 minute incubation at 37° C. in 4 mL of 0.1 M (Na⁺) citrate bufferpH 7.3 with an addition of 0.05 mL of 20 mM TM-PEG.

For Examples 4 and 5 were used: suspended fixed endothelial cells, towhich biotin was conjugated. These were obtained by incubating HUVEC inone 23 cm² cell culture flask for 30 minutes at 37° C. with 2 mL of 21mM Hepes buffer pH 8.2 with 105 mM sodium citrate and 2 mM CaCl₂ with anaddition of 0.05 mL of TM-PEG and 0.05 mL of 2 mg/mL of NHS-biotin. Thecells were detached with a small cell rubber policeman. The so suspendedcells were fixed for 30 minutes at room temperature by the addition of 4mL of STF fixation solution (se below) and washed by three fold repeatedcentrifugation and re-suspension in 0.02 M Hepes buffer pH 7.5 with 0.1M NaCl. Suspended, fixed, biotin conjugated HUVEC were immobilized onthe surface of streptavidin-treated micro test plate wells (Labsystemsproduct 95029290) by the addition to each well of about 4,000 cellssuspended in 0.1 mL of 0.02 M Hepes buffer with 0.1 M NaCl and incubatedfor 18 hours at room temperature.

For Examples 2 and 3, HUVEC cultured in micro test plate wells wereused. They were washed two times in PBS and devitalized by addition of0.1 mL of 0.1% formalin in PBS per well and incubated for 10 minutes atroom temperature with subsequent twice repeated wash with 0.2 mL PBS.

Samples of blood plasma were obtained from the Laboratory of ClinicalChemistry, University Hospital of Linkoping with generous support of Dr.Tomas Lindahl. Samples with known APTT- and PTK-values were obtainedwithin 4 hours of routine analysis with the reagents and instrumentsused by the laboratory during June 1994. The same laboratory alsogenerously supplied citrate anti-coagulated samples from individualswithout known haemostatic disorders, i.e. from healthy (normal)individuals. These samples from patients and normal individuals werestored at -70° C. for a maximum of 5 months prior to use.

Reagents for the determination of D-dimer (a degradation product offibrin) with ELISA technique, 1241 TintElize D-dimer, and latextechnique, 150707 Minutex D-dimer, were obtained from Biopool AB, Umea,Sweden.

tPA, tissue plasminogen activator with single-chain structure,single-chain tPA, product 122101 from Biopool AB, was dissolved in 0.5 M(Na⁺) Hepes-buffer pH 8.5 to a concentration of 3,000 IU/mL.

Hirudin, product 53000, was obtained from American Diagnostics Inc.,Greenwich, Conn. Hirudin was dissolved in H₂ O to a concentration of 100ATu/mL.

Heparin, 100 IU/mL for injectional use, was obtained from L.oslashed.vens kemiske fabrik, Ballerup, Denmark.

STF (Streck Tissue Fixative) containing diazolidinyl-urea,2-bromo-2-nitro-propane-1,3-diol, zinc sulphate and citrate, wasobtained as ready to use solution from Streck Laboratories Inc., Omaha,Nebr., USA.

TM-PEG (metoxypolyethylene-glycol-tresylate), product M-3038, wasobtained from Sigma Chemical Company, St. Louis, Mo., USA.

NHS-LC-biotin (sulphosuccinimidyl-6-(biotiamido)hexanoate), product21335, was obtained from Pierce, Rockford, Ill., USA.

NaCl, CaCl₂, Hepes, citric acid, formalin and 6-amino-hexanoic acid wereof high commercial quality. Water of high purity was obtained from anapparatus employing ion-exchange and reversed osmosis serviced by theDepartment of Clinical Chemistry, University Hospital Linkoping.

Example 1

The invention was practised for analysis of coagulative activity with aprotocol resembling a one-step prothrombin analysis according to Quick,wherein 1 volume of citrate anti-coagulated plasma was mixed with onevolume of thromboplastin reagent containing CaCl₂ and registration ofcoagulation time was performed.

This experiment utilized a commercial Quick reagent, 50220Thromboplastin from Biopool AB, Umea, composed of a suspended extractfrom delipidated rabbit brain tissue. A corresponding reagent accordingto the invention was HUVEC reagent 1, which contained 0.2×10⁶ cells/mLof suspended fixed HUVEC in 50 mM Hepes buffer pH 7.3 containing 15 mMCaCl₂, 30 g/L PEG 6000, 30 g/L BSA and 2 g/L Triton X-100. The analyseswere performed with a coagulation instrument with photometric detectionof coagulum formation, Coagulator 4, Behnk Elektronik, with 0.15 mL ofsample and 0.15 mL of reagent, both temperature equilibrated at 37° C.

The following citrate anti-coagulated samples were analyzed:

Pooled plasma from normal individuals (NP) and NP diluted 1:2 (NP50%)and NP 1:4 (NP25%) with a barbiturate buffer with citrate and NaCl(Tampon pour SPA, Diagnostica Stago, Franconville, France). Moreover thefollowing were analyzed: a plasma sample with a PTK-value below 25%(PTK<25%) and a plasma sample (TRBP) from a thrombotic patient. Theplasma sample PTK<25% was pooled from individuals treated with vitamin-Kantagonists who according to analysis performed during April-May 1994 bythe Laboratory for Clinical Chemistry, University Hospital Linkoping,showed PTK-values of less than 25%. The results of the analysis withQuick-reagent and HUVEC-reagent 1 are depicted in the table below, wherethe results have been placed in order of precedence according to theircoagulation times and with the coagulation time in seconds given withinparenthesis.

    ______________________________________                                        Quick-reagent          HUVEC-reagent 1                                        ______________________________________                                        NP          (12.9)     TRBP       (214.1)                                       TRBP (13.4) NP (233.7)                                                        NP50% (17.1) NP50% (282.6)                                                    PTK<25% (23.1) NP25% (383.2)                                                  NP25% (25.6) PTK<25% (459.4)                                                ______________________________________                                    

It may especially be noted that TRBP has a longer coagulation time thanNP in analysis with the Quick-reagent, while TRBP has a shortercoagulation time than NP with analysis according to the invention,HUVEC-reagent 1.

A plasma sample from a thrombophiliac thus displays a shortenedcoagulation time in analysis according to the invention. This is not thecase with conventional analysis. Diluted normal plasma as well as pooledplasma with long coagulation times according to other conventionalmethod (PTK) display prolonged coagulation ) times both with analysisaccording to the invention and with conventional Quick-methodology.However, analysis according to the invention seems to more clearlydistinguish PTK<25% than is the case with the Quick-reagent. Tosummarize, in the displayed experiment, analysis according to the.invention gives more relevant diagnostic information about thrombophiliathan does conventional analysis, and at least as good information onhaemophilia.

For purposes of confirmation, the same thrombotic plasma (TRBP) and thesame pool of normal plasma (NP) were analyzed on a later occasion, andthen with another HUVEC-reagent and another commercial preparation ofthromboplastin reagent (IL Test PT-fibrinogen, product 97567-10,Instrumentation Laboratories, Milano, Italy). The analysis according tothe invention was performed with HUVEC-reagent 2 and HUVEC-reagent 3,which differed only in that they contained 0.23×10⁶ cells and 0.46×10⁶cells/mL, respectively. The experiment with HUVEC-reagent 3 wasperformed in duplicate in the same instrument channel. Otherexperimental details were identical to those detailed above. Thefollowing results were obtained with the coagulation times denoted inseconds.

    ______________________________________                                        Quick-reagent  HUVEC-reagent 2                                                                            HUVEC-reagent 3                                   ______________________________________                                        NP    12.3         381.0        436.8 and 438.9                                 TRBP 12.6 277.5 208.4 and 236.6                                             ______________________________________                                    

Hereby confirmation was obtained, that coagulation analysis according tothe invention can differentiate the plasma from a thrombotic patientfrom that of a normal individual in a case where conventional analysisis without diagnostic effect.

Example 2

Quantitative analysis of coagulative activity according to the inventionis here applied on citrate anti-coagulated plasma from 10 patientsreferred to the Department of Clinical Chemistry, University HospitalLinkoping, for investigation concerning cause of thrombotic disease andon the same type of samples from 10 healthy individuals, i.e.individuals who have never been afflicted with thrombotic disease.

In the analysis, a sample of size 0.10 mL was added to a micro testplate well, the bottom surface of which was covered with a confluentlayer of fixed HUVEC. The reaction was started by addition of 0.010 mLof 0.5 M CaCl₂ and allowed to proceed minutes at room temperature underagitation on a shaking apparatus with circular eccentric movement, 1 mmeccentricity and 200 revolutions per minute. The reaction was stopped byaddition of 0.010 mL of 20 ATu/mL of hirudin. The wells were emptied oftheir contents and washed three times with 0.25 mL of 0.15 M NaCI.Cell-associated fibrin was dissolved with 0.1 mL of human serum with 300IU/mL of tPA which was allowed to act for 20 minutes at room temperatureafter which the amount of D-dimer in the serum was determined by ELISA.The following results, presented in order of precedence according toincreasing content of D-dimer, were obtained:

    ______________________________________                                        Thrombotic patients                                                                          Healthy individuals                                              D-dimer (ng/mL) D-dimer (ng/mL)                                             ______________________________________                                         75             73                                                               77  78                                                                        77  88                                                                        83  89                                                                        215  99                                                                       366 119                                                                       577 179                                                                       952 193                                                                      1849 467                                                                      2553 470                                                                    ______________________________________                                    

Four of the 10 thrombotic patients displayed higher, three considerablyhigher, amounts of fibrin associated with endothelial cells than any ofthe healthy individuals, when blood plasma from these were analyzedaccording to the invention. Statistical analysis also showed that thegroups most probably differed from each other p<0.05 according toone-tailed t-test. Analysis according to the invention of coagulativeactivity according to this example displays some of the diagnosticpossibilities of the procedure.

Example 3

Quantitative analysis of coagulative activity according to the inventionwith the same protocol as in Example 2 was performed on seven bloodplasma samples anti-coagulated with citrate. These samples had beenanalyzed less than 2 hours earlier on May 18 1994 according to PTK andAPTT by the Laboratory of Clinical Chemistry, University HospitalLinkoping. The results for these seven samples are shown below in orderof precedence according to their PTK-times. PTK-values in percent aregiven within parentheses. D-dimer according to the invention.

    ______________________________________                                        PTK (s)       APTT (s) D-dimer (ng/mL)                                        ______________________________________                                        19.4 (145)    35.3     368                                                      24.1  (91) 45.3 296                                                           25.0  (84) 31.7 334                                                           29.7  (59) 39.7 198                                                           31.0  (54) 47.3 195                                                           44.9  (29) 33.4 149                                                           59.1  (20) 48.5 130                                                         ______________________________________                                    

Linear regression analysis shows that coagulation activity according tothe invention shows that good negative correlation with PTK-times andsimilar good positive correlation with PTK-values in percent. A negativecorrelation with APTT-times is also evident

This result indicates that coagulation analysis according to theinvention can find use in diagnostic contexts where PTK- andAPTT-analyses are used, i.e. in the identification of individuals withbleeding tendencies and in guiding and monitoring of substitutiontherapy for haemophiliacs and therapy for thrombosis prophylaxis withoral anti-coagulants (warfarin) or subcutaneous anti-thromboticum(heparin).

The correlation between analysis according to the invention and PTK andAPTT are both better than that between PTK and APTT. This high-lightsthe over-all, global, character of the analysis according to theinvention.

Example 4

Quantitative analysis of coagulative activity according to theinvention, but with a protocol that differs in several details from thatof Example 3, was applied to blood plasma anti-coagulated with citratefrom four thrombotic patients and one healthy individual. According tothis protocol, 0.10 mL of plasma was added to micro test plate wellscoated with streptavidin with attached devitalized fixedbiotin-conjugated endothelial cells or, as control, to similar wells butwithout cells. The coagulation reaction was initiated by addition of0.025 mL of 0.15 M CaCl₂ and was stopped by 0.025 mL of 100 IU/mLheparin. The amount of fibrin formed was determined quantitatively byaddition of 0.025 mL of 3000 IU/mL of tPA, which under 20 minutes wasallowed to process formed fibrin into soluble degradation products.These were measured as D-dimer by ELISA.

The results of the analysis, D-dimer in ng/mL, are presented below,where the value for the well containing endothelial cells is denoted"with HUVEC" and the control well "without HUVEC". The difference isgiven as "with-wihout":

    ______________________________________                                        with HUVEC       without HUVEC                                                                             with-without                                     ______________________________________                                        Thrombotic                                                                             188         142          46                                            patient 1                                                                     Thrombotic  933 140  853                                                      patient 2                                                                     Thrombotic 2836 174 2662                                                      patient 3                                                                     Thrombotic 3597  52 3545                                                      patient 4                                                                     Healthy 2487 227 2260                                                         individual                                                                  ______________________________________                                    

In this example two of the four thrombotic patients (patient 3 andpatient 4) displayed greater fibrin deposit on the endothelial cellsthat the healthy individual. Thrombotic patient 1 displayed markedly lowD-dimer development although fibrin deposition was verifiedmicroscopically. Perhaps this is a thrombotic patient with reducedability to degrade fibrin in spite of high levels of plasminogenactivator.

Example 5

Quantitative analysis of fibrinolytic activity according to theinvention was demonstrated with a series of five blood plasma samplesfrom a healthy individual, to which small volumes of concentrated tPAsolution was added so that the final addition of tPA activity was 0,0.73, 2.9, 11.7, and 46.7 IU/mL Of these tPA enriched plasmas, 0.10 mLwas added to micro test plate wells with attached endothelial cells ofthe same kind as was used in Example 4. A limited amount of fibrin wasformed during 7 minutes upon addition of 0.025 mL of CaCl₂.

The fibrin formation was stopped by addition of 0.025 mL of 100 IU/mL ofheparin, after which the fibrinolytic reactions were allowed to continuefor an additional 60 minutes. The amount of fibrin degradation productsthus formed was used as a measure of the fibrinolyfic activity and wasdetermined as D-dimer in the well contents by ELISA. The results werethe following:

    ______________________________________                                        Sample     added tPA (IU/mL)                                                                          D-dimer (ng/mL)                                       ______________________________________                                        A          0             41                                                     B 0.73  42                                                                    C 2.9  45                                                                     D 11.7  317                                                                   E 47 3640                                                                   ______________________________________                                    

The example shows that analysis according to the invention can be usedfor analysis of fibrinolytic activity. Plasma with high levels of tPAdisplay high levels of D-dimer.

What is claimed is:
 1. A method of diagnosing thrombophilia in a mammalcomprising: contacting, in vitro, a sample of blood or plasma of anindividual suspected of having a thrombophilic disorder with fixedendothelial cells or outer membranes of fixeda) determining the rate ofcoagulum formation or the time required for the formation of apredetermined amount of coagulum, comparing the rate or time with areference value from blood or blood plasma obtained from normalindividuals, wherein if the rate is faster or the coagulation time isshorter, the sample is assessed to come from an individual havingdisposition for thrombosis or b) halting the coagulation, determiningthe amount of coagulum, and comparing the amount of coagulum withreference values of amounts of coagulum obtained from normalindividuals, wherein if the amount of coagulum is greater than thereference values the sample is assessed to come from an individualhaving disposition for thrombosis, or c) halting the coagulation,allowing coagulum dissolving reactions to continue, determining theamount of dissolved coagulum, comparing the amount of dissolved coagulumwith reference values of dissolved coagulum obtained from normalindividuals, wherein if the dissolved amount of coagulum is smaller thanthe reference values the sample is assessed to come from an individualhaving disposition for thrombosis, or d) halting the coagulation,allowing coagulum dissolving reactions to continue, determining theamount of remaining coagulum, comparing the amount of remaining coagulumwith reference values of remaining coagulum obtained from normalindividuals, wherein if the amount of remaining coagulum is greater thanthe reference values the sample is assessed to come from an individualhaving disposition for thrombosis.
 2. The method according to claim 1,wherein said sample of blood or blood plasma is prior to the contactingwith fixed endothelial cells or outer membranes of fixed endothelialcells reversibly anticoagulated, and returned to a coagulable state atsaid contacting in vitro.
 3. The method according to claim 2 whereinsaid sample of blood or blood plasma is anticoagulated with citrate orEDTA that binds Ca²⁺ ions, and returned to coagulable state by additionof Ca²⁺ ions.
 4. The method according to claim 2, wherein said sample ofsaid blood or blood plasma is anticoagulated with acoagulation-inhibiting substance selected from hirudin or heparin, andthat said return to coagulable state is accomplished by addition ofantibodies which neutralize the coagulation-inhibiting activity of saidhirudin, or by addition of heparinase to neutralize thecoagulation-inhibiting activity of said heparin.
 5. The method accordingto claim 1, wherein the determination is performed on a quantity ofblood or blood plasma of 0.01-1.0 ML.
 6. The method according to claim1, wherein, during the determination, the coagulation is halted by theaddition of a coagulation-inhibiting substance, which is selected fromthe group consisting of hirudin and heparin.
 7. The method according toclaim 1, wherein the determination is performed with afibrinolysis-inhibiting amount of a fibrinolysis-inhibiting substanceadded to the reaction mixture, which substance is selected from thegroup of 6-amino-hexanoic acid and antibodies against tPA and uPA. 8.The method according to claim 1, wherein the coagulum is detected bymeans of changes in optical or rheological properties of the reactionmixture, including changes in light transmission or viscosity.
 9. Themethod according to claim 1, wherein the coagulum is detected bymeasuring some component or degradation product of the coagulum,selected from fibrin, thrombocytes, or soluble degradation products offibrin.
 10. The method according to claim 9, wherein the amount ofthrombocytes in the coagulum is measured after enzymatic degradation ofthe coagulum.
 11. The method according to claim 1, wherein the amount offibrin is measured via its enzymatic degradation with plasmin, tosoluble degradation products, which are measured with immunologicaltechnique.
 12. The method according to claim 11, wherein saiddegradation products are measured with immunological technique specificfor D-dimer fragments.
 13. The method of claim 1 wherein said mammal isa human.